Method for providing a beverage additive

ABSTRACT

Methods are described that provide controlled release of beneficial bacteria to the oral cavity. Such bacteria can subsequently colonize the oral cavity and displace or suppress the growth of pathogenic microorganisms. Described devices include a stabilized preparation of such beneficial bacteria and provide a consumable liquid, wet foam, or gel that includes such bacteria when used.

This application is a divisional application of U.S. patent applicationSer. No. 16/036,789 filed on Jul. 16, 2018. These and all otherreferenced extrinsic materials are incorporated herein by reference intheir entirety. Where a definition or use of a term in a reference thatis incorporated by reference is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein is deemed to be controlling.

FIELD OF THE INVENTION

The field of the invention is beverage additives, more specificallybeverage additives that include bacteria.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Oral disease, such as dental caries, is a significant health care issue,compounded by the prevalence of sugar and other carbohydrate-basedsweeteners in the modern diet. In addition, stained teeth arecosmetically unappealing.

Tooth surfaces are absorbent and can become stained or discolored by theuse of tobacco products, eating or drinking certain foods and beverages(e.g., coffee, tea and red wine), the buildup of dental plaque, theprocess of aging, diseases, trauma, medications, congenital conditions,and other environmental effects. Teeth are comprised of an inner dentinlayer, an outer enamel layer and an acquired pellicle. The acquiredpellicle is a proteinaceous layer derived from saliva that forms on thesurface of tooth enamel.

Regular brushing with a dentifrice and regular dental care are effectivein treating and reducing the prevalence of oral disease and improvingtheir appearance. Extrinsic and intrinsic staining of the teeth canoccur. Extrinsic staining is staining of the acquired pellicle that canoccur when compounds such as tannins and polyphenolic compounds come incontact with teeth during eating, drinking or smoking. These compoundsthen become trapped in and tightly bound to the proteinaceous layer onthe surface of the teeth. Extrinsic staining can be removed bymechanical methods of tooth cleaning, such as brushing or flossing andby chemical cleaning methods. Even with regular brushing and flossing,rapid or slow accumulation can develop into noticeable intrinsic toothdiscoloration. Intrinsic staining can be caused by staining compoundsthat penetrate the enamel layer and the dentin layer or can arise fromsources within the tooth. Intrinsic staining is difficult to remove andcannot typically be removed by mechanical methods of tooth cleaning, buthigh chemical concentrations and/or prolonged chemical cleaning methodscan be used to remove some or all of this type of staining.

White, unstained teeth are considered cosmetically desirable. Teeth canbe whitened by, for example, mechanical cleaning methods, veneers thatare placed over the teeth, and chemical bleaching.

While tooth whitening products are known in the art, these products aretraditionally used by those seeking the cosmetic benefit of whiterteeth. However, there is a different population of consumers who desirewhiter teeth and/or more oral benefits including cleaner teeth,healthier gums, and decreased oral malodor. Therefore, there is a desireto provide oral hygiene products that can deliver oral care benefits inaddition to tooth whitening. Furthermore, oral hygiene time is typicallylimited and so there is a desire to deliver these oral care benefitsquickly and conveniently as part of a daily oral hygiene regimen. As aresult less intrusive treatment modalities have been pursued. Amongthese are the use of bacteria that can inhabit the oral cavity but donot cause oral disease. Such bacteria can, for example, releasecompounds that improve or prevent an oral disease or condition (such asperoxide), or can colonize the oral cavity and displace disease-causingbacteria. Effective delivery of such bacteria in an acceptable manner,however, remains challenging.

Various means for introducing beneficial bacteria into humans have beenproposed, however to date these have been directed towards directingrelatively robust bacteria to the human gut (i.e. stomach, smallintestine, and/or large intestine). Such approaches emphasize rapidpassage through the mouth and upper portions of the digestive tract inorder to preserve viability, and as such do not provide sufficientlyslow release for colonization of the mouth. In addition, labile bacteriaare not suitable for such applications. Australian Patent ApplicationPublication No. 2008317000, to Moore, describes treatment of obesity byadministration of a Bacteroides bacteria, with such administrationintended to introduce the bacteria to the gut of the person beingtreated. All publications identified herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.Although use of a liquid carrier is mentioned it is not clear how thiswould be accomplished. United States Patent Application Publication No.2007/0098847, to Teissier, describes the use of dehydrated andgranulated “lactic bacteria” that has been coated with a solid vegetablefat in foods, which provides protection from moisture and release in thegut. While the application mentions introduction of such coated granulesinto liquids it is not clear how such granules would be subsequentlyconsumed, as they would presumably separate out from the solution.

European Patent Application No. 1020123, to Vesely and Milano, describedbeverages that include a mixture of three different “lactic bacteria”species, which are prepared by adding a lyophilized bacterialpreparation to the beverage immediately prior to consumption. Thelyophilized bacteria can be provided in a “plug” for the containerholding the beverage, which is opened and the contents mixed with thebeverage prior to consumption. Similarly, U.S. Pat. No. 5,895,648, toVesely et al, describes the use of a pair of containers, one of whichcontains a consumable liquid, cream or paste and the other of whichcontains a lyophilized mixture of bacteria, wherein the container thatholds the lyophilized bacteria is opened and mixed with the consumablematerial at the moment of consumption. U.S. Pat. No. 5,170,888, toGoncalves, describes a container with two compartments that containcomponents to be mixed prior to use and are separated by a membrane. Thecontainer includes a rotating blade that advances along a screw threadwhen turned in order to pierce the membrane and allow mixing of thecomponents. U.S. Pat. No. 6,105,760, to Mollstam and Casas, describe asimilar device, and additionally described using a detached drinkingstraw to pierce such a membrane. EP Patent Application No. 2341008, toBiogaia et al, describes a device for mixing a moisture sensitivecomponent with a liquid component immediately prior to use. The moisturesensitive component is sealed within a portion of a cap behind afrangible barrier. The cap also includes a piston device that locks intotwo positions; movement between the two positions forces the end of thepiston through the frangible barrier to release the moisture sensitivecomponent into the liquid. It should be appreciated, however, that suchmethods and devices are intended for the delivery of probiotic mixturesto the gut. As such they dispense the entire volume of dehydrated orlyophilized bacteria into the liquid volume, where they are consumed asa dilute and uniform suspension.

Thus, there is still a need for a convenient and acceptable way todeliver bacteria to the oral cavity of a person.

SUMMARY OF THE INVENTION

The inventive subject matter provides apparatus, systems and methods inwhich a stabilized probiotic bacteria composition is introduced into theoral cavity of an individual at concentrations and for a period of timethat permits at least transient colonization of surfaces of the oralcavity, for example the surfaces of the teeth and/or gumS. The probioticbacterial composition is provided as a component of a probioticbeverage. As defined herein, the term beverage is inclusive of aconsumable liquid, wet foam, gel, or other fluid substance. In apreferred embodiment the probiotic bacterial composition is provided ina stabilized form (for example, an anhydrous form) that is mixed with anaqueous component by a user at or immediately prior to the time ofconsumption to form the probiotic beverage.

One embodiment of the inventive concept is beverage additive thatincludes an isolated, non-pathogenic, hydrogen peroxide bacterialspecies or strain and a genetically modified LDH-deficient bacterialstrain and a metabolizable carbon source (e.g. a carbohydrate), wherethe bacterial formulation is or includes a stabilized bacteriapreparation (for example, a lyophilized bacterial population, bacteriain an edible oil suspension, etc.). Such a stabilized bacteriapreparation can also include a stabilizing agent. Suitable isolated,non-pathogenic, hydrogen peroxide-producing bacterial species or strainsinclude Lactobacillus, Bifidobacteria, viridans Streptococcus,Leuconostoc, Pediococcus, and LactococcuS. Suitable genetically modifiedLDH-deficient bacterial strains include a genetically modified strain ofStreptococcus mutans.

Another embodiment of the inventive concept is beverage additive devicethat includes a beverage additive, which in turn includes an isolated,non-pathogenic, hydrogen peroxide bacterial species or strain and anLDH-deficient bacterial strain. The isolated, non-pathogenic, hydrogenperoxide bacterial species or strain and the LDH-deficient bacterialstrain are provided as a stabilized bacteria preparation. The beverageadditive device also includes a moisture resistant barrier that enclosesthe beverage additive, along with a disrupting mechanism positioned tocircumvent the moisture resistant barrier (for example, by displacing,rupturing, or piercing the barrier) and place the stabilized probioticbacterial preparation in contact with a consumable aqueous solution. Insome embodiments the moisture resistant barrier includes a packet thatencloses the beverage additive, where the packet is essentiallyimpermeable to water. In some embodiments the moisture resistant barrieris a submergible body, and can be positioned within an aperture or alumen or passage of the beverage container. Such a submergible body caninclude a wall, an interior volume that encloses the bacterialformulation, and a through hole that traverses the wall. In someembodiments the beverage additive device is incorporated into a beveragecontainer. In other embodiments the beverage additive device isconfigured to reversibly couple to a beverage container.

Another embodiment of the inventive concept is a beverage additivedevice that includes a body with an interior flow channel, a loweraperture, and an upper aperture. The beverage additive device alsoincludes an isolated, non-pathogenic, hydrogen peroxide bacterialspecies or strain and an LDH-deficient bacterial strain, where theisolated, non-pathogenic, hydrogen peroxide bacterial species or strainand the LDH-deficient bacterial strain are provided as a stabilizedbacteria preparation. The stabilized bacteria preparation is attached oradhered to at least a portion of an inner wall of the flow channel ofthe beverage additive device. In some embodiments a porous membrane(which can includes one or more pores) is positioned between thestabilized bacteria preparation and an interior space of the flowchannel. In such embodiments at least a portion of the one or more poresare occupied by a water soluble compound. Such a beverage additivedevice can be configured to fit through an opening in a beveragecontainer, for example as a drinking straw. In some embodiments thebeverage additive device is provided in packaging that includes adessicant.

Another embodiment of the inventive concept is a fluid dispensing devicethat includes a first reservoir containing an isolated, non-pathogenic,hydrogen peroxide bacterial species or strain and an LDH-deficientbacterial strain, where the isolated, non-pathogenic, hydrogen peroxidebacterial species or strain and the LDH-deficient bacterial strain areprovided as a stabilized bacteria preparation (for example, as asuspension in an edible oil). The fluid dispensing device also includesa second fluid reservoir the contains a fluid vehicle. First and secondflow channels are in fluid communication with the first and second fluidreservoirs, respectively, and with a valve. In some embodiments thefirst and second flow channels are separate channels that couple to thevalve at distinct and different Positions. In other embodiments thefirst and second flow channels are segments of a common flow channelthat couples to the valve. A dispensing channel (which can includemixing features) is in fluid communication with the valve, which in turnprovides fluid communication between the first and second reservoirswhen in the open position. Either or both of the first and second fluidreservoirs can be pressurized, for example by containing liquids thatincorporate a dissolved gas. The fluid contents of either or both of thefirst and second fluid reservoirs can also include additional componentsthat aid in providing the desired form or function of the final fluidproduct produced by the device, such as a surfactant and/or apharmaceutical compound.

Another embodiment of the inventive concept is a method of introducing aprobiotic formulation to an oral cavity, by providing a beverageadditive device that incorporates a probiotic beverage additive, amoisture resistant barrier that encloses the beverage additive, and adisrupting mechanism that is placed or positioned to circumvent themoisture resistant barrier and place the stabilized probiotic bacterialpreparation in contact with an aqueous solution. The disruptionmechanism is activated to generate a consumable beverage, gel, or wetfoam, and the consumable beverage or wet foam in contact with the oralcavity for a period of time sufficient for the bacteria of the probioticbeverage additive to adsorb or otherwise adhere to a surface of the oralcavity. The probiotic beverage additive includes an isolated,non-pathogenic, hydrogen peroxide bacterial species or strain and anLDH-deficient bacterial strain in the form of a stabilized bacteriapreparation. In some embodiments the method includes a step ofreversibly coupling the beverage additive device to a beverage containerenclosing the consumable aqueous solution. In some embodiments thedisrupting mechanism includes a valve, and the method further includes astep of opening the valve.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a beverage container of the inventive concept.

FIG. 2 shows an alternative beverage container of the inventive concept.

FIG. 3 shows another alternative beverage container of the inventiveconcept.

FIGS. 4A and 4B depict a device of the inventive concept that couples toa beverage container reversibly. FIG. 4A shows a cross section of aexample of such a device, with a housing that includes threadscomplementary to threads of a beverage container. FIG. 4B depicts adevice as shown in FIG. 4A engaged with a beverage container having athreaded opening.

FIG. 5 depicts an embodiments of the inventive concept in which aprobiotic bacterial preparation is placed within a flow channel of adevice used for drinking liquids.

FIG. 6 depicts and embodiment of the inventive concept that utilizesmaterials stored in separate reservoirs that are mixed upon use togenerate a consumable beverage, wet foam, or gel.

DETAILED DESCRIPTION

Embodiments of the inventive concept provide methods and devices thatdispense preparations of bacteria that can occupy or colonize the oralcavity of a user on consumption of a beverage. These bacteria can beselected displace or suppress the growth of bacteria responsible forvarious oral conditions, such as dental caries, xerostomia, and/orhalitosis. Such bacterial preparations can be relatively labile, beingintolerant of the human digestive tract and becoming non-viable rapidlyon liquid suspension if not provided with a suitable surface forcolonization. In some embodiments a stabilized (for example, by drying,lyophilization, and/or the use of a preservative compound) preparationof orally-colonizing bacteria is provided in a sealed container,envelope, capsule, or similar enclosure that is pierced or otherwiseaccessed immediately prior to beverage consumption. In some embodimentssuch access results in dispensing of the entire or nearly (e.g. greaterthan 70%) volume of the stabilized probiotic bacterial suspension into abeverage to be consumed. In other embodiments a significant fraction(e.g. greater than about 30% of the stabilized probiotic bacterialpreparation) is retained in the accessed enclosure following the initialpiercing action, and is released from the enclosure gradually onbeverage consumption. In such an embodiment the orally colonizingbacteria retained in the accessed enclosure can show extended viabilityrelative to those in free suspension in the bulk beverage. Similarly, insuch an embodiment orally colonizing bacteria can be released atrelatively high concentration from the accessed enclosure on taking thebeverage into the mouth, the high concentration of bacteria improvingthe efficiency of colonization of oral surfaces (e.g. teeth, mucosa,etc.) of a user. One should appreciate that embodiments of the inventiveconcept provide a simple and attractive way to introduce beneficialbacteria into the oral cavity.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value with a range is incorporated into the specification asif it were individually recited herein. All methods described herein canbe performed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

As used herein, and unless the context dictates otherwise, the term“coupled to” is intended to include both direct coupling (in which twoelements that are coupled to each other contact each other) and indirectcoupling (in which at least one additional element is located betweenthe two elements). Therefore, the terms “coupled to” and “coupled with”are used synonymously.

The instant invention provides methods and compositions for theintroduction of beneficial probiotic bacteria to the oral cavity, whichcan be useful in maintenance of oral health (for example, toothwhitening). A composition of the invention includes one or moreisolated, non-pathogenic, hydrogen peroxide-producing species or strainsof bacteria, and optionally, LDH-deficient mutans Streptococcus. Certainbacteria can produce hydrogen peroxide at a level that can whiten teeth,and potentially effect a desired change in appearance and/or structureof a tooth surface. Examples of appearance and structural changesinclude, but are not necessarily limited to, whitening, stain bleaching,stain removal, plaque removal, and tartar removal. Furthermore,colonization of the oral cavity by beneficial bacteria can provideadditional oral care benefits such as reducing the incidence of dentalcaries, reducing the incidence of periodontitis, reducing the incidenceof oral bacterial infections, reducing the incidence of yeast or fungalovergrowth, reducing the incidence and/or severity of halitosis,reducing the incidence or severity of xerostomia, promotion of woundhealing, or a combination of such effects.

The invention provides compositions and methods for improving oralhealth and/or whitening tooth surfaces using a composition that includesa mixture of probiotic bacteria that includes one or morenon-pathogenic, hydrogen peroxide-producing viridans Streptococcispecies or strains, and/or one or more non-pathogenic, hydrogenperoxide-producing Lactobacillus species or strains and/or one or morenon-pathogenic, hydrogen peroxide-producing Bifidobacteria species orstrains and/or one or more non-pathogenic, hydrogen peroxide producingLactococcus species or strains and/or one or more non-pathogenic,hydrogen peroxide producing Pediococcus species or strains and/or one ormore non-pathogenic, hydrogen peroxide producing Leuconostoc species orstrains.

In one embodiment of the invention the bacterial strains can begenerally recognized as safe (GRAS), and can transiently attach, adhere,and/or adsorb to a tooth surface by virtue of electrostaticinteractions, van der Waals interactions, or protein or polysaccharideadhesins on the bacterial surface that recognize and interact withmolecules present on the tooth surface.

Examples of viridans Streptococci species include, but are not limitedto S. sanguis, S. parasanguis, S. gordonii, S. oralis, S. uberis, S.mitis, S. rattus, S. salivariaus, S. vestibularis, S. angionosus, S.constellatus, S. intermedius, S. mutans, S. sobrinus, S. milleri, S.cricetus, and S. mitior. Examples of Lactobacillus species include, butare not limited to, L. acidophilus, L. jensenii, L. catenaforme, L.leichrnanni, L. plantarum, L. johnsonii, L. gasseri, L. delbrueckii, L.casei, L. brevis, L. salivarius, L. gasseri, L. sobrius, L. rhamnosus,L. reuteri, L. fermentum, L. paracasei, L. dextranicurn, and L.helveticus. Examples of Bifidobacteria species include, but are notlimited to B. angulatum, B. animalis, B. asteroides, B. bifidum, B.boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B.cuniculi, B. dentium, B. gallicum, B. gallinarum, B indicum, B. longum,B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B.pseudolongum, B. psychraerophilum, B. pullorum, B. ruminantium, B.saeculare, B. scardovii, B. simiae, B. subtile, B. thermacidophilum, B.thermophilum, and B. urinali. Examples of other non-pathogenic bacteriathat can produce hydrogen peroxide include, without limitation,Pediococcus species, such as P. acidilactici, Leuconostoc species, suchas L. mesenteroides, Lactococcus species such as L. lactis.

The quantity of hydrogen peroxide produced by bacteria can beexperimentally determined. See e.g. Hillman and Shivers, Arch. Oral.Biol., 33:395-401 (1988). The culture liquor of cells grown in thepresence of oxygen is incubated with 40 μg/ml horseradish peroxidase and0.4 μmol/ml o-dianisidine. After 2 minutes, the reaction is stopped bythe addition of 0.02 ml of 5N HCL. The optical density of the sample ismeasured at 410 nm and the hydrogen peroxide concentration of the sampleis calculated from a standard curve prepared using authentic hydrogenperoxide and an extinction coefficient at 230 nm of 81M⁻¹cm⁻¹. In oneembodiment of the invention the bacteria can produce at least about 0.5,1, 2, 5 mM or more of H₂O₂ or any range or value between about 0.1 andabout 5 mM.

In one embodiment of the invention a composition of the inventionincludes one or more isolated Streptococcus oralis strains and/or one ormore S. uberis strains. Compositions of the invention can optionallycomprise one or more isolated strains or species of mutans streptococcusthat are LDH-deficient. Colonization of the oral cavity with such acombination of non-pathogenic, hydrogen peroxide-producing bacteriaand/or mutans streptococcus provides a significant practical advantagein that the colonization of the oral cavity with such a combination canreduce the incidence of dental caries, reduce the incidence ofperiodontitis, reduce the incidence of oral bacterial infections, reducehealing time for oral wounds, reduce the incidence of Candida and/orfungal overgrowth, reduce the incidence and/or severity of xerostomia,and can reduce discoloration of tooth enamel.

Streptococcus oralis (previously known as S. sanguis Type II) and S.uberis are important components in maintaining the normal, healthybalance of microorganisms that comprise the periodontal flora. SeeSocransky et al., Oral MicrobioL. ImmunoL. 3:1-7 (1988); Hillman andShivers, Arch. OraL. Biol., 33:395-401 (1988); Hillman et al., Arch.OraL. Biol., 30:791-795 (1985). S. oralis can also be found in dentalplaque and has been demonstrated to correlate with periodontal health,in particular by interfering with the colonization by periodontalpathogens such as Aggregetobacter actinomycetemcomitans, Porphyromonasgingivalis, Peptostreptococcus micros, and Campylobacter rectus.Compositions of the invention can comprise one or more isolated strainsof S. oralis, for example, ATCC 35037, ATCC 55229, ATCC 700233, ATCC700234 and ATCC 9811. Other strains of S. oralis include KJ3 and KJ3sm.KJ3sm is a naturally occurring genetic variant of KJ3 that is resistantto 1 mg/ml streptomycin. The streptomycin resistance is advantageousbecause it provides a marker for easy isolation of the bacteria.Additionally, streptomycin resistant strains are slightly attenuated anddo not survive as long in an oral cavity as wild-type strains. Thisproperty is useful where the goal is to non-persistently colonize theoral cavity of an animal with the probiotic bacteria.

S. uberis can also be found in dental plaque and has been demonstratedto correlate with periodontal health, in particular by interfering withthe colonization by periodontal pathogens such as Tannerellaforsythensis, P. micros, C. rectus, and Prevotella melaninogenica.Compositions of the invention can comprise one or more isolated strainsof S. uberis, for example, ATCC 13386, ATCC 13387, ATCC 19435, ATCC27958, ATCC 35648, ATCC 700407, ATCC 9927, strain KJ2 or strain KJ2sm.KJ2sm is a naturally occurring genetic variant of KJ2 that is resistantto 1 mg/ml streptomycin and provides the same advantages as forstreptomycin-resistant strains of S. oralis. One or more isolatedstrains of S. oralis or one or more isolated strains of S. uberis, orboth, can be used in compositions and methods of the invention.

Compositions of the invention can include a mixture of probioticbacteria that includes one or more isolated mutans streptococcusbacteria species deficient in the production of lactic acid. Thesespecies include, for example, S. rattus, S. cricetus, S. mutans, S.sobrinus, S. downeii, S. macacae, and S. ferus. A mutans streptococcusof the invention does not substantially produce L(+) lactatedehydrogenase (LDH). Such a strain is termed an LDH-deficient strain. AnLDH-deficient strain of mutans streptococcus produces 75%, 80%, 90%,95%, 98%, 99%, or 100% less lactic acid than wild-type strains of mutansstreptococcus. An LDH-deficient mutans streptococcus strain can be anaturally occurring strain of mutans streptococcus or a geneticallymodified strain of mutans streptococcus. LDH-deficient mutansstreptococcus can compete with and/or displace pathogenic bacteria suchas S. mutans, a principal etiological agent of dental caries, in theoral cavity. LDH-deficient mutans streptococcus stains will compete withS. mutans for the same nutrients, colonization sites, etc. Therefore,colonization of the oral cavity with LDH-deficient mutans streptococcusstrains is associated with prevention and/or reduction of the incidenceof dental caries. LDH-deficient strains of mutans streptococcus arenon-pathogenic, alter the microenvironment of the oral cavity to preventcolonization or outgrowth of pathogenic organisms, and/or displacepathogenic organisms from the oral cavity where the pathogen is part ofthe host's indigenous flora.

Examples of LDH-deficient mutans streptococcus strains include, forexample, S. rattus JH145 (ATCC 31377) (a spontaneous,naturally-occurring LDH-deficient mutant) and JH140 (ATCC 31341) (achemically-modified LDH-deficient mutant). See e.g., Stanshenko &Hillman, Microflora of plaque in rats following infection with anLDH-deficient mutant of Streptococcus rattus, Caries Res. 23:375-377(1989); Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:Isolation and preliminary characterization. Infect. Immun. 21:206-212(1978); see also Abhyankar et al., Serotype c Streptococcus mutansmutatable to lactate dehydrogenase deficiency. J. Dent. Res. 64:1267-71(1985).

An LDH-deficient strain of mutans streptococcus can be derived from amutans streptococcus strain using, for example, chemical or physicalmutagenesis techniques. Strains that are mutagenized using thesetechniques are considered genetically modified strains. For example, amutans streptococcus strain can be subjected to mutagens such as nitrousacid, formic acid, sodium bisulphate, UV light, base analog mutagens,including for example, 5-bromo-deoxyuridine (5BU), alkylators such asethyl methane sulfonate (EMS), methyl methane sulfonate (MMS),diethylsulfate (DES), and nitrosoguanidine (NTG, NG, MNNG). See e.g., InVitro Mutagenesis Protocols, Braman, Ed., Humana Press, 2002.

Naturally-occurring, spontaneous LDH-deficient mutans streptococcusstrains can be prepared using methods disclosed in, for example,Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:isolation and preliminary characterization. Infect. Immun. 21:206-212(1978). Spontaneous LDH-deficient mutants occur at the rate ofapproximately 10⁻⁵ frequency. See Johnson et al., Cariogenic potentialin vitro in man and in vivo in the rat of lactate dehydrogenase mutantsof Streptococcus mutans. Arch. Oral Biol. 25:707-713 (1980).

Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be differentiated from LDH-producing strains of mutansstreptococcus by plating the bacteria on glucose tetrazolium medium.LDH-deficient mutans streptococcus colonies will be bright red andrelatively larger in size than colonies of the parent strain, which arewhite and relatively smaller in size on the glucose tetrazolium medium.Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be used in a composition of the invention.

An LDH-deficient strain of S. rattus has been isolated. Briefly, aculture of S. rattus BHT-2 was grown overnight to saturation in ToddHewitt broth, and diluted samples were spread on glucose tetrazoliummedium to give approximately 300 colonies per plate. Wild-type, acidproducing colonies are white on this medium. LDH-deficient mutants arebright red. S. rattus JH145 was one red colony amid approximately100,000 white colonies that were screened. S. rattus JH145 is thereforea naturally-occurring, LDH-deficient mutant.

LDH-deficient strains of mutans streptococcus, such as LDH-deficientmutants of S. rattus BHT-2, produce less total titratable acid whenincubated in the presence of glucose and other sugars or polyols, makesubstantially less lactic acid when incubated in the presence of glucosein the case of resting and growing cultures, adhere better tohydroxyapatite and accumulate more plaque when grown in the presence ofsucrose. LDH activity can be assayed as described by Brown &Wittenberger (J. BacterioL. 110:604, 1972).

Terminal pH can be determined by subculturing strains (1:100) inTodd-Hewitt broth containing 1% glucose. After 48 hours incubation incandle jars at 370 C, the absorbance at 580 nm and pH of the culturescan be determined. Lactic acid concentration of cultures can bedetermined by gas-liquid chromatography. See Salanitro & Muirhead,Quantitative method for the gas chromatographic analysis of short-chainmonocarboxylic and dicarboxylic acids in fermentation media. Appl.Microbiol. 29:374-381 (1975); Hillman et al., Acetoin production bywild-type strains and a lactate dehydrogenase-deficient mutant ofStreptococcus mutans. Infect. Immun. 55:1399-1402 (1987). Additionally,any genetic modification techniques known to those of skill in the artcan be used to create an LDH-deficient mutans streptococcus strain froman LDH-producing mutans streptococcus parent strain. For example, an LDHgene or a portion of an LDH gene can be deleted or mutagenized,including, for example, insertional mutagenesis techniques. Othermutagenesis techniques include, for example, homologous recombination,recursive sequence recombination, oligonucleotide-directed mutagenesis,site-directed mutagenesis, error-prone PCR, phosphothioate-modified DNAmutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,site saturation mutagenesis, ensemble mutagenesis, recursive ensemblemutagenesis, and chimeric nucleic acid creation. Therefore, any geneticmodification technique that disables an LDH gene can be used to producean LDH-deficient mutans streptococcus strain. In one embodiment of theinvention, the LDH-deficient strains, whether naturally-occurring orgenetically-modified mutants, have a reversion frequency less than 10⁻⁷and produce less than about 10% of the parental level of lactatedehydrogenase activity.

The use of two or more different species of bacteria can provide anadvantage over using a single species. This is because different speciesof bacteria colonize different surfaces or portions of teeth. Therefore,the use of more than one species of bacteria can be used to “blanket”all or most surfaces of the teeth, whereas the use of only one speciesof bacteria may result in certain surfaces or portions of the teethbeing uncolonized. Therefore, all surfaces of the teeth are exposed towhitening action.

Compositions of the invention can further comprise one or more carbonsources that are metabolizable by the one or more isolated,non-pathogenic, hydrogen peroxide-producing bacterial species or strainsor the one or more lactate dehydrogenase deficient mutans Streptococcusspecies or strains or both types of species or strains. Carbons sourcesinclude, but are not limited to, for example, glucose, sorbitol,mannitol, fructose, galactose, maltose, sucrose, xylose, lactose,glycerol or combinations thereof.

The compositions of the invention can comprise a pharmaceuticallyacceptable or nutritionally acceptable carrier. The carrier isphysiologically compatible with the oral cavity of the subject to whichit is administered. Carriers can be comprised of solid-based, drymaterials for formulation into tablet, capsule, lozenge, or powderedform. A carrier can also be comprised of liquid or gel-based materialsfor formulations into liquid, gel, and chewing gum forms. Suitableliquid or gel-based carriers include but are not limited to: water,physiological salt solutions, urea, alcohols and derivatives, andglycols (e.g., ethylene glycol or propylene glycol). The composition ofthe carrier can be varied so long as it does not interfere significantlywith the therapeutic activity of the bacterial strains of the invention.

A composition can be formulated to be suitable for oral administrationin a variety of ways, for example in a semi-solid, liquid (including,e.g., a viscous liquid, a paste, a gel, or a solution), a dried mass, adentifrice, a mouth wash, an oral rinse, a liquid suspension, a topicalagent, a paste, a gel, a solid food, an oral rinse, and the like. Otherformulations will be readily apparent to one skilled in the art. Acomposition of the invention can include a nutrient supplement componentand can include any of a variety of nutritional agents, as are wellknown, including vitamins, minerals, essential and non-essential aminoacids, carbohydrates, lipids, foodstuffs, dietary supplements, and thelike.

Compositions of the invention can also include natural or syntheticflavorings and food-quality coloring agents, all of which are compatiblewith maintaining viability of the bacterial species or strains of theinvention.

A composition of the invention can include one or more gelling agents.The concentration of the gelling agent may be greater than about 2, 4,6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80 or less than about 80, 70, 60,50, 40, 30, or 20 percent by weight of the composition.

Suitable gelling agents and adhesion agents useful in the presentinvention include, for example, silicone, polyethylene oxide, polyvinylalcohol, poly alkyl vinyl ether-maleic acid copolymer (PVM/MA copolymer)such as, Gantrez AN 119, AN 139, and S-97, polyvinyl alcohol,polyacrylic acid, Poloxamer 407 (Pluronic), polyvinyl pyrrolidone-vinylacetate copolymer (PVP/VA copolymer), such as Luviskol VA, and PlasdoneS PVP/VA, polyvinyl pyrrolidone (PVP, e.g., K-15 to K-120),Polyquaterium-11 (Gafquat 755N), Polyquaterium-39 (Merquat plus 3330),carbomer or carboxypolymethylene (Carbopol), hydroxy propyl methylcellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, cornstarch, carboxymethyl cellulose, gelatin and alginate salt such assodium alginate, natural gums such as gum karaya, xanthan gum, Guar gum,gum arabic, gum tragacanth, and mixtures thereof.

A humectant or plasticizer can be present in compositions of theinvention. Humectants or plasticizers include, for example, glycerin,glycerol, sorbitol, polyethylene glycol, propylene glycol, and otheredible polyhydric alcohols. The humectants or plasticizers can bepresent between at about 1% to about 99%, about 10% to about 95%, or atbetween about 50% and about 80% (or any range between 1% and 99%) byweight of a composition.

Bacteria of the invention can be prepared in, for example, a fermenter.The bacteria can be harvested from the fermenter and can be, forexample, concentrated. Bacteria of the invention can be prepared for useby, for example, dehydration or spray drying. Spray drying generallycomprises spraying a suspension of bacteria in a vessel and under asteam of hot air. Bacteria can also be prepared for use bymicroencapsulation (see e.g., U.S. Pat. No. 6,251,478), freeze-drying,or by coating with a protective substance such as, for example, lipidmaterial such as triacylglycerols, waxes, organic esters, soybean oil,cottonseed oil, palm kernel oil, and esters of long-chain fatty acidsand alcohols.

Methods of Whitening Teeth

The invention provides methods for delivering a composition thatprovides one or more oral care benefits, including tooth whitening, tothe surfaces of the oral cavity comprising applying a composition of theinvention to the teeth and/or adjacent soft tissue of a subject.

The bacterial species or strains can be present in a composition of theinvention in a therapeutically effective amount. Therapeuticallyeffective means effective to alleviate, reduce, prevent and/orameliorate one or more symptoms of dental caries, periodontitis,bacterial infections or diseases, oral wounds, Candida or fungalovergrowth, halitosis, or xerostomia-induced dental caries orperiodontal disease or to alleviate, reduce, prevent, or amelioratestains or discoloration on the teeth either permanently or temporarily.Therapeutically effective also means effective to promote wound healingin an oral cavity.

A therapeutically effective amount is an amount of a composition of theinvention at high enough levels to provide effective colonization of theoral cavity, but low enough to avoid serious side effects (at areasonable benefit/risk ratio). The therapeutically effective amount ofa composition of the invention may vary with the condition of anindividual's oral cavity, the age and physical condition of theindividual being treated, the severity of any pathologies within theoral cavity, the duration of treatment, the nature of any concurrenttherapy, the specific form of the source employed, and the particularvehicle from which the composition is applied.

The compositions of the invention can be applied in a therapeuticallyeffective amount to the mucosal tissue of the oral cavity, to thegingival tissue of the oral cavity, to the surface of the teeth and/orany combination thereof for the treatment and/or prevention of stainedand/or discolored teeth. A composition of the invention may be swallowedor may rinsed around the oral cavity and spit out.

The bacterial strains of the invention can form at least a part of thetransient or indigenous flora of an oral cavity and exhibit additionalbeneficial prophylactic and/or therapeutic effects in the cavity. Suchexposure and/or colonization of the oral cavity can have a beneficialeffect on dental caries, periodontitis (including, for example,early-onset periodontitis, localized and generalized juvenileperiodontitis, and rapidly progressive and adult periodontitis), oralbacterial infections and diseases, oral wounds, Candida or fungalovergrowth, halitosis, or xerostomia-induced dental caries, woundhealing, or a combination thereof.

Compositions can be administered to an oral cavity of a subject such asan animal, including a mammal, for example, a human, a non-humanprimate, a dog, a cat, a rat, a mouse, a horse, a goat, or a rabbit.

One embodiment of the invention provides a method of non-persistentlycolonizing an oral cavity of a subject with therapeutically-effectivebacteria comprising administering to the oral cavity of a subject acomposition of the invention. In one embodiment of the invention theadministered bacterial strains do not permanently colonize the oralcavity, rather the strains are present in the oral cavity for about 1day, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 3months or about 12 months after administration of the bacteria.

Compositions of the invention can be administered at a dose of about1×10³, 1×10⁵, 1×10⁷, 1×10⁹, or 1×10¹¹ CFU (or any range or value betweenabout 1×10³ and about 1×10¹¹) of viable bacteria. A dose of acomposition of the invention can be administered at three times a day,twice a day, once a day, every other day, two times a week, weekly,biweekly, or monthly. One, two, or more doses of a composition of theinvention can be administered per day for about 1 week, about 2 weeks,about 1 month, about 2 months, about 3 months, about a year or more.

Compositions of the invention can be used daily as part of an oral careregimen. Using compositions of the invention as part of a daily oralcare regimen allows a user to achieve and sustain a variety of desiredoral care benefits, including but not limited to white, tartar-freeteeth.

A composition of the invention can be applied to the teeth and/or softtissue for between about 1 minute and about 8 hours. In someembodiments, the composition can be applied for greater than about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210,240, 270, 300, 330, 360, 390, 420, 450, 480 minute(s) (or any range orvalue between about 1 and about 480 minute(s)) and/or less than 480,450, 420, 390, 360, 330, 300, 270, 240, 210, 180, 150, 120, 90, 60, 50,40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute(s) (or any rangeor value between about 480 and about 1 minute(s)) and any combinationthereof, wherein the bacterial species or strains have a concentrationbetween about 0.01% and about 50%, or about 0.1% to about 25%, or about1.0% to about 10% or any ranges or values in between 0.01% and 50% byweight of the composition. Such a regimen could be advantageously usedonce a day for greater than about one month, two months, four months,six months, twelve months, eighteen months, two years, five years, eightyears, ten years and/or less than about fifteen years, ten years, eightyears, five years, two years, 18 months, 12 months, six months, fourmonths, two months, one month and any combination thereof. In anotherembodiment such a regimen could be advantageously used once or twice aday for greater than about one month and less than about 5 years.

A kit of the invention can contain a one month, two month, three month,four month, five month, six month, or 12 month supply of a compositionof the invention. A composition of the invention can be packaged and, inturn, a plurality of the packaged compositions can be provided in astorage container or outer package or carton.

Embodiments of the inventive concept include devices and compositionsthat provide controlled release of beneficial probiotic bacteria asdescribed above (which can be relatively labile), which in turn releasesthe bacteria into the oral cavity during consumption of a beverage innumbers and over a period of time sufficient to encourage colonizationof oral surfaces. Examples of such devices and compositions are shown inFIGS. 1 to 3 .

FIG. 1 shows an embodiment of the inventive concept in which astabilized probiotic bacterial preparation (as described above) isincorporated into a delivery system that instills the bacterialpreparation into a liquid that is consumed by a user. In such anembodiment a dried or otherwise stabilized probiotic bacterialpreparation 110 is provided in a moisture resistant or water-proofenclosure 100. Suitable moisture resistant or water-proof enclosuresinclude a foil and/or plastic lined pouch, packet, or sachet. In someembodiments the moisture resistant or water proof enclosure canincorporate a portion of the walls of an associated beverage container140, for example by providing moisture resistant or water-proof barriersthat are fixed to an inner wall of the beverage container and thatenclose a dried or otherwise stabilized probiotic bacterial preparationwithin an intervening space. A puncturing tool 120 is provided that canbe used by a user to puncture or rupture the moisture resistant orwater-proof enclosure 100 in order to release the probiotic bacterialpreparation 110 into a beverage. The puncturing tool 120 can be attachedto the beverage container 140, for example by one or more toolsupport(s) 130 that align the puncturing tool with the moistureresistant or water-proof enclosure. The tool support 130 can beperforated or constructed of a relatively weak or thin material in orderto provide sufficient mechanical strength to support the puncturing tool120 during manufacturing and shipping, but to be fragile enough to allowa user to crush or deform the tool support upon use. In some embodimentsthe puncturing tool 120 can be secured using a string, line, tether, orsimilarly flexible attachment that allows a user to grasp the puncturingtool and move it into position to puncture or rupture the moistureresistant or water-proof enclosure.

In some embodiments the enclosure 100 can be retained in a liquid flowpath of the beverage container, and the puncturing tool 120 dimensionedto provide one or more through-holes that provide retention of about 30%or more of the volume of probiotic bacterial preparation within theenclosure 100 following puncture or rupture. Such a through-hole can befrom 100 μm to 5 mm in diameter, and the number of through-holesprovided on use of the puncturing tool 120 can range from 1 to 100 ormore. Towards that end the puncturing tool 120 can be provided with oneor more shafts with a maximum diameter of 100 μm to 5 mm. In suchembodiments the retained bacteria can be released into the oral cavityof the user on swallowing a portion of the beverage. In otherembodiments rupture of the enclosure 100 can release essentially all ofthe bacteria (e.g. about 70% or more) into a beverage held within theenclosure to form a relatively homogenous bacterial suspension.

In order to use such a delivery system a user can utilize the puncturingtool 120 to pierce or otherwise rupture the moisture resistant orwater-proof enclosure and release the stabilized probiotic bacterialpreparation. On release at least a portion of the stabilized probioticbacterial preparation can fall into a beverage contained in the lowerportion of the beverage container 140. In some embodiments at least aportion (for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, or more than about 99%) of the stabilized probioticbacterial preparation is retained in the punctured enclosure followingpuncturing, and is released into the oral cavity of a user on flow ofthe beverage through the remains of the enclosure on consumption.

In other embodiments the stabilized probiotic bacterial preparation canbe provided in a filter or similarly porous envelope or container thatis positioned to avoid puncture or rupture by the puncturing tool whenin use. Such a filter or similarly porous envelope can retain all orsome of the bacterial mass within the fluid flow path as the beverage isconsumed. In some embodiments such a filter or similarly porous envelopecan be displaced into the interior of the beverage container followingrupture of the enclosure by the puncturing tool, and subsequentlyrelease bacteria into the beverage over a period of time (for example,30 seconds to 30 minutes). Such an embodiment advantageously retainslabile bacteria that may be sensitive to the beverage's composition overtime in a relatively protected environment, and provide a continuoussupply of active bacteria to the oral cavity on consumption of thebeverage.

In another embodiment of the inventive concept the moisture resistant orwater-proof enclosure can be configured as a closure 100A of thebeverage container 140, as shown in FIG. 2 . In such an embodiment theclosure 100A can include a friction surface for gripping an internalwall of the beverage container 140, for example in a neck portion of thebeverage container, as is typical for a bottle closure or stopper. Theclosure 100A can enclose the dried or otherwise stabilized probioticbacterial preparation 110, and can include one or more a piercable orfrangible wall(s). These can support and/or surround the bacterialpreparation 110. A puncturing tool 120 is provided that can bepositioned to puncture or rupture the piercable or frangible wall of theclosure 100A. As shown, the puncturing tool 120 can be incorporated intoor traverse a portion of the closure 100A, and supported by one or moretool support(s) as described above. Alternatively, the puncturing toolcan be provided on a tether, line, or other flexible support that isattached to the beverage container 140 and permits a user to maneuverthe puncturing tool into position.

In some embodiments the enclosure 100A can include a through-holethrough which the beverage can be consumed, and can be retained orreplaced in a liquid flow path of the beverage container, and thepuncturing tool 120 dimensioned to provide one or more through-holesthat provide retention of about 30% or more of the volume of bacterialpreparation within the enclosure 100A following puncture or rupture. Insuch embodiments the retained bacteria can be released into the oralcavity of the user on swallowing a portion of the beverage. In otherembodiments rupture of the enclosure 100A can release essentially all ofthe bacteria (e.g. about 70% or more) into a beverage held within theenclosure to form a relatively homogenous bacterial suspension. In suchan embodiment the enclosure 100A can be removed and/or discardedfollowing puncture or rupture.

In order to use such a delivery system a user can utilize the puncturingtool 120 to pierce or otherwise rupture the moisture resistant orwater-proof enclosure 100A and release the stabilized probioticbacterial preparation. On release at least a portion of the stabilizedprobiotic bacterial preparation can fall into a beverage contained inthe lower portion of the beverage container 140. In some embodiments atleast a portion of the stabilized probiotic bacterial preparation isretained in the punctured enclosure, and can optionally be released intothe beverage by manipulation of the beverage container 140 by the user(for example, by shaking, rocking, and/or inverting the container.

In another embodiment of the inventive concept, shown in FIG. 3 , themoisture resistant or water-proof enclosure is provided as a capsule100B that encloses a dried or otherwise stabilized probiotic bacterialpreparation 110. The capsule 100B can, for example, lie within a neck,lumen, or other extension of an associated beverage container 140. Insome embodiments the capsule is affixed to an interior wall of thebeverage container 140, and acts prevent release of its contents priorto consumption. In some of such embodiments the capsule 100B is affixedin a temporary manner, and can be dislodged as described below, topermit consumption of a contained beverage. Once dislodged such acapsule 100B can be retained in this portion of the beverage containerby one or more stops. As shown, an upper stop 160A and a lower stop 160Bcan be provided to retain the capsule 100B with the beverage containerand prevent premature release of the bacterial preparation 110,respectively. Such a stop can, for example, be configured as acontinuous or discontinuous ring or collar, as one or more rod(s),bump(s) or similar projections into the neck or lumen, or as a net ormesh. Such a stop can be formed from the material of the beveragecontainer, or can be provided as an added appliance that is coupled tothe beverage container. In some embodiments the lower stop 160B can bepliant, such that the capsule 100B inserts into the body of the beveragecontainer 140 on use, and can be retained therein.

As shown, a dispensing tool 120A is provided. In some embodiments thedispensing tool 120 can include a terminus, point, projection, or othercutting or puncturing portion that can create one or more opening(s) inthe capsule 100B when wielded by a user. This permits the beverage toenter the punctured s capsule and release at least a portion of thebacterial preparation into the beverage on consumption. In otherembodiments the dispensing tool 120A is configured to simply dislodgethe capsule, thereby permitting flow of the beverage around thedislodged capsule. In such an embodiment the capsule can include one ormore aperture(s) 150, which permit release of at least a portion of thebacterial preparation into the beverage on consumption. If an apertureis present in the capsule the beverage container 140 can be providedwith a moisture resistant or water-proof seal that encloses the open endof the neck of the beverage container prior to use. In such embodimentsthe aperture 150 can be oriented such that it is not exposed to thebeverage prior to dislodging of the capsule 100B.

In some embodiments the enclosure 100B can be retained in a liquid flowpath of the beverage container, and the puncturing tool 120 dimensionedto provide one or more through-holes that provide retention of about 30%or more of the volume of bacterial preparation within the enclosure 100Bfollowing puncture or rupture. In such embodiments the retained bacteriacan be released into the oral cavity of the user on swallowing a portionof the beverage. In other embodiments rupture of the enclosure 100B canrelease essentially all of the bacteria (e.g., about 70% or more) into abeverage held within the enclosure to form a relatively homogenousbacterial suspension.

Another embodiment of the inventive concept is a dispensing device thatcan be attached to a pre-existing commercial beverage container, forexample bottle water, a soft drink, etc. An example of such anembodiment is shown in FIG. 4A. The device 400 includes an enclosure 410that provides a water/moisture barrier that surrounds a stabilizedprobiotic bacterial preparation as described above. The enclosure can beplaced within a housing 440 that interfaces with the beverage container.Such a housing can, for example, be dimensioned to slip over the neck ofsuch a container, insert into the neck or opening of such a beveragecontainer, and/or include threads that engage complementary threads ofsuch a beverage container. As shown in FIG. 4B, in use the device 400 isreversibly coupled to a beverage container 450 via such a housing. Thehousing 440 can be coupled to a guide 405, through which passes adispensing tool 420. Such a dispensing tool can include a projection,blade, point, or similar device that is positioned to rupture orotherwise disrupt the water/moisture barrier when it is depressed. Insome embodiments the dispensing tool can be held in a pre-dispenseposition by a temporary support 430. Such a temporary support can beremoved by a user, or crushed or similarly deformed by a user throughthe application of downwards pressure on the dispensing tool 420 inorder to dispense the stabilized probiotic composition into a beverageheld within the beverage container.

Another embodiment of the inventive concept is a device that can beinserted into a liquid beverage, and through which a user appliessuction to consume the beverage through a channel or lumen of thedevice. An example of such a device is shown in FIG. 5 . As shown, thedevice 500 can be configured as an elongated tube or straw, with a wall510, a channel or lumen 520 through which liquid passes when in use, alower aperture 530 that is submerged in a beverage while in use, and anupper aperture 540 that is placed in or at the entrance of the oralcavity when in use. The device includes an insert that containsstabilized probiotic bacteria 550 as described above. The stabilizedprobiotic bacteria are adhered to the wall of the channel or lumen 520,and in some embodiments can be at least partially covered by a porouswall or membrane 560, which is in turn affixed to the wall of the lumen520. The pores of the porous wall or membrane 560 can be occupied by awater-soluble filler (such as a water soluble polymer) that preventsmoisture from reaching the stabilized probiotic bacteria prior to use.Alternatively, in embodiments where no porous wall or membrane ispresent or where the pores are left unoccupied the device 500 suppliedin a water resistant container or envelope (for example, an envelope orcontainer that includes a dessicant). If present, a porous wall ormembrane can include a vent feature 570 that permits some flow of fluidfrom the lower aperture 530 to the upper aperture 540 when suction isapplied to the upper aperture. In use the lower aperture 530 is insertedinto a beverage to be consumed and suction applied by the user to theupper aperture 540. Movement of the liquid beverage through the lumendislodges the stabilized probiotic material for transport to the oralcavity. In embodiments that include a porous wall or membrane in whichthe pores have been filled such liquid would initially dissolve thewater-soluble material from the pores and subsequently release thestabilized probiotic bacteria through the pores and into the oralcavity.

Another embodiment of the inventive concept is a device that provides aconsumable and flowable suspension, emulsion, gel, and/or wet foam bymixing the contents of a fluid reservoir that includes stabilizedprobiotic bacteria and a second fluid reservoir that includes a vehicle.One or both of such reservoirs can be pressurized (for example, withnitrogen, nitrous oxide, air, etc.) to provide a motive force for movingthe contents of these reservoirs through fluid flow channels, and forcombining the contents of the reservoirs at or near a dispensing nozzle.An example of such an embodiment is shown in FIG. 6 . As shown, such adevice 600 includes a first reservoir 610, which includes stabilizedprobiotic bacteria. Such stabilized bacteria are provided in a liquid orother flowable media, for example an edible oil. The device 600 alsoincludes a second reservoir 620, which includes a flowable vehicle, forexample an aqueous solution. Such an aqueous solution can includecompounds that aid in forming the suspension, emulsion, gel, and/or wetfoam, for example surfactants, wet foam stabilizers, thickeners, etc.Either or both of the reservoirs 610, 620 can include additionalcompounds that are useful in combination with the probiotic bacteria,including carbohydrates, flavoring agents, coloring agents, mineralsthat support or reinforce tooth enamel, and/or pharmaceutical compounds.

As shown in FIG. 6 , the reservoirs 610, 620 each have an associatedflow channel (630 and 640, respectively) through which reservoircontents flow when the device is activated. The driving force for suchflow can be provided by a pressurized or dissolved gas, which can beeither included in a reservoir or placed in fluid communication with it.It some embodiments both reservoirs are pressurized and dispensing ofreservoir contents occurs through individual flow channels associatedwith each reservoir. In other embodiments only one of the reservoirs ispressurized, and flow is provided by placing both reservoirs along acommon flow channel. Dispensing of the contents of the reservoirs can beactivated by opening a valve 650, which opens communication between theflow channels 630, 640 and a dispensing channel 660. The dispensingchannel 660 can include mixing features to improve blending of thecontents of the reservoirs, for example projections into the interiordispensing channel, configuration of some or all of the dispensingchannel as a tortuous path, and variations in the cross section of thedispensing channel.

Mixing of reservoir contents in the dispensing channel 660 provides aconsumable and flowable material that incorporates the probioticbacteria. The nature of the consumable and flowable material is at leastpartially dependent on the contents of the reservoirs 610, 620. Forexample, blending of an edible oil suspension of probiotic bacteria inreservoir 610 with an aqueous solution in reservoir 620 can provide anoil-in-water emulsion. Inclusion of a surfactant in one or bothreservoirs can provide a micellar suspension. Inclusion of a dissolvedgas in one or both of the reservoirs can provide a wet foam. Inclusionof a gel-forming polymer (such as an alginate) in one reservoir and anactivating component (such as a calcium salt) in the remaining reservoircan provide a gel.

One embodiment of the invention provides a method of non-persistentlycolonizing an oral cavity of a subject with a mixture of probioticbacteria that includes bacteria which do not produce (or produce atreduced levels relative to wild type bacteria) acids that can degradetooth enamel. In a typical method a composition of the invention isadministered to the oral cavity of a subject, for example as describedabove. In one embodiment of the invention the administered bacterialstrains do not permanently colonize the oral cavity, rather the strainsare present in the oral cavity for about 1 day, about 1 week, about 2weeks, about 3 weeks, about 1 month, about 3 months or about 12 monthsafter administration of the bacteria. Without wishing to be bound bytheory, the Inventor believes that such colonization can at leasttransiently reduce the population of potentially harmful wild typebacteria with the oral cavity.

Compositions of the invention can be administered at a dose of about1×10³, 1×10⁵, 1×10⁷, 1×10⁹, or 1×10¹¹ CFU (or any range or value betweenabout 1×10³ and about 1×10¹¹) of viable bacteria. A dose of acomposition of the invention can be administered at three times a day,twice a day, once a day, every other day, two times a week, weekly,biweekly, or monthly. One, two, or more doses of a composition of theinvention can be administered per day for about 1 week, about 2 weeks,about 1 month, about 2 months, about 3 months, about a year or more.

Compositions of the invention can be used as part of an oral careregimen. Using compositions of the invention as part of a daily oralcare regimen allows a user to achieve and sustain a variety of desiredoral care benefits, including but not limited to white, tartar-freeteeth.

A kit of the invention can contain a one week, two week, one month, twomonth, three month, four month, five month, six month, or 12 monthsupply of a composition of the invention, and also can includeinstructions for use. The composition within the kit can be individuallypackaged as distinct dose units. Alternatively, a plurality of thepackaged compositions can be provided in a storage container or outerpackage or carton. In some embodiments a kit of the inventive conceptcan include features or components that improve stability of theproduct, for example a dessicant.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of introducing a probiotic bacterialformulation to an oral cavity of an individual, comprising; providing abeverage container, comprising: a flow path providing flow of fluidcontents of the beverage container out from the beverage container; aprobiotic bacterial composition comprising an isolated, non-pathogenic,hydrogen peroxide bacterial species or strain and an LDH-deficientbacterial strain, wherein the isolated, non-pathogenic, hydrogenperoxide bacterial species or strain and the LDH-deficient bacterialstrain are provided as a stabilized bacteria preparation; a passageencompassing a submergible body comprising a moisture resistant barrierthat encloses the beverage additive, wherein the passage comprises anupper stop and a lower stop positioned to retain the submergible bodywithin the flow path, wherein the submergible body is movable betweenthe upper and lower stops; and a dispensing mechanism positioned tocircumvent the moisture resistant barrier and place the stabilizedbacteria preparation in contact with fluid contents of the beveragecontainer on passage of fluid contents of the beverage container throughthe flow path; activating the dispensing mechanism; and dispensing amixture of the stabilized bacteria preparation and fluid contents of thebeverage container to the oral cavity.
 2. The method of claim 1, whereinthe moisture resistant barrier comprises a packet enclosing the beverageadditive, and wherein the packet is essentially impermeable to water. 3.The method of claim 1, wherein the dispensing mechanism is reversiblycoupled to the beverage container.
 4. The method of claim 1, wherein thedispensing mechanism comprises a puncturing device positioned to contactthe submergible body on use, and wherein activation comprises puncturingthe submersible body with the puncturing device.
 5. The method of claim4, wherein the puncturing device is coupled to a plunger, and whereinactivation comprises depressing the plunger.